Supplemental Materials

Estrogen -α in Medial Amygdala Neurons Regulates Body Weight

Pingwen Xu1,9, Xuehong Cao1,9, Yanlin He1, Liangru Zhu1,2, Yongjie Yang1, Kenji Saito1, Chunmei Wang1, Xiaofeng Yan1, Antentor Othrell Hinton Jr.1, Fang Zou1, Hongfang Ding1, Yan Xia1, Chunling Yan1, Gang Shu1, San-Pin Wu3, Bin Yang4, Yuxin Feng5, Deborah J. Clegg6, Richard DeMarchi4, Sohaib A. Khan7, Sophia Y. Tsai3, Francesco J. DeMayo3, Qi Wu1, Qingchun Tong8, Yong Xu1, 3*

1Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; 2Department of Gastroenterology , Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People's Republic of China; 3Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; 4Department of Chemistry, Indiana University, Bloomington, IN 47405;5Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 6Department of Biomedical Research, Diabetes and Obesity Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; 7Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati, College of Medicine, Cincinnati, OH 45267; 8Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030.

9These authors equally contributed to this work.

*Correspondence should be addressed to: 1100 Bates Street, Rm 8070, Houston, Texas 77030. E-mail: [email protected], Telephone: (713)-798-7199, Fax: (713)-798-7187.

Running Title: MeA ERα regulates physical activity & body weight

1 Supplemental Figure 1. Validation of selective deletion of ERα in SIM1 neurons in SIM1- ERα-KO mice.

(A-B) Dual immunofluorescence for GFP (green) and ERα (red) in the MeA in SIM1- Cre/Rosa26-GFP mice (control, A) and in ERαlox/lox/SIM1-Cre/Rosa26-GFP mice (SIM1-ERα- KO, B). Left panel: ERα immunoreactivity; middle panel: GFP immunoreactivity; right panel: merged images. Arrows point to double-labeled neurons. MeA, medial amygdala; opt, optic tract. Scale bars = 100 µm.

(C-D) Numbers of SIM1 (GFP) neurons, ERα neurons, and double-labeled neurons in the MeA in female (A) and male (B) SIM1-Cre/Rosa26-GFP or ERαlox/lox/SIM1-Cre/Rosa26-GFP mice. Data are presented as mean±SEM. N=3 in each group.

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Supplemental Figure 2. expression in the amygdala and hypothalamus.

(A-B) Relative mRNA levels of indicated in the MeA from chow-fed male (A) or female (B) control and SIM1-ERα-KO mice. Data are presented as mean±SEM. N=6 in each group. (C-D) Relative mRNA levels of indicated genes in the hypothalamus from chow-fed male (C) or female (E) control and SIM1-ERα-KO mice. Data are presented as mean±SEM. N=6 in each group.

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Supplemental Figure 3. Open field tests. Anxiety-related responses in open field tests in male and female control and SIM1-ERα-KO mice (8 weeks of age). (A-B) Distance travelled in male (A) and (B) mice. (C) Time spent in the center region of the open field. (D) Numbers of fecal boli. N=7-12/group. Results are shown as MEAN ± SEM.

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Supplemental Figure 4. Light-dark tests. Anxiety-related responses in light-dark tests in male and female control and SIM1-ERα-KO mice (8 weeks of age). (A) Time spent in the light chamber. (B) Latency to enter the dark chamber. (C) The total number of transitions between the light and dark chambers. (D) Ratio of time spent in the light chamber vs the dark chamber. N=7- 12/group. Results are shown as MEAN ± SEM.

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Supplemental Figure 5. Elevated plus maze tests. Anxiety-like behavior measured in male and female control and SIM1-ERα-KO mice (8 weeks of age). (A) Open time ratio. (B) Open entry ratio. N=7-12/group. Results are shown as MEAN ± SEM.

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Supplemental Figure 6. Reproductive functions in female SIM1-ERα-KO mice. (A) Appearance of vaginal opening. Data are presented as mean±SEM. N=18 in each group. (B) Appearance of the first estrus. Data are presented as mean±SEM. N=16 or 18 in each group. (C) Length of diestrus, proestrus and estrus relative to the entire estrous cycles. Data are presented as mean±SEM. N=14 or 20 in each group. (D) Percentage of mice that successfully delivered pups. Data are presented as mean±SEM. N=8 or 20 in each group. (E) Averaged litter size. Data are presented as mean±SEM. N=20 or 6 in each group. (F) Averaged time period between mating day and birth day of pups. Data are presented as mean±SEM. N=18 or 6 in each group.

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Supplemental Figure 7. Metabolic profiles of PVN-ERα-KO mice.

(A) Post hoc visualization of GFP in the PVN in an ERαlox/lox mouse receiving AAV-Cre-GFP stereotaxic injections. 3V, 3rd ventricle; PVN, paraventricular nucleus of the hypothalamus. The scale bar = 200 µm.

(B) Gains in body weight after virus injections. Note mice were fed with regular chow for the first 7 days and then with HFD for the rest period. Data are presented as mean±SEM. N=4 in each group. * P<0.05 in two-way ANOVA analysis followed by post hoc Bonferroni tests.

(C) Cumulative food intake. Data are presented as mean±SEM. N=4 in each group. * P<0.05 in two-way ANOVA analysis followed by post hoc Bonferroni tests.

(D) Feeding efficiency calculated as the ratio between changes in body weight (B) and cumulative food intake (C).

(E) 24-hour physical activity. (F) Sum physical activity during the light or dark cycles. Data are presented as mean±SEM. N=4 in each group.

(G) 24-hour body temperature. (H) Averaged body temperature during the light or dark cycles. Data are presented as mean±SEM. N=4 in each group.

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Supplemental Figure 8. Co-localization of glutamate and SIM1 in the MeA. Immunofluorescence for glutamate (green in A), TOMATO red fluorescence (B), and the merge (C) in the MeA in SIM1-Cre/Rosa26:tdTOMATO mice. Arrows point to double-labeled neurons. MeA, medial amygdala; opt, optic tract. Scale bars = 50 µm.

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Supplemental Figure 9. Co-staining of GABA and SIM1 in the MeA. Immunofluorescence for GABA (green in A), TOMATO red fluorescence (B), and the merge (C) in the MeA in SIM1-Cre/Rosa26:tdTOMATO mice. Note that there is no double-labelled neuron. MeA, medial amygdala; opt, optic tract. Scale bars = 50 µm.

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Supplemental Figure 10. Anterograde tracing of MeA SIM1 neurons. (A-B) Injection tract showing accurate stereotaxic delivery of Ad-IN/W virus into the MeA of SIM-Cre mice. (C-F) Immunofluorescence for WGA in the Red N (C-D), DRN (E-F) and MRN (E). Aq, aqueduct; DRN, dorsal Raphe nuclei; MeA, medial amygdala; MRN, median Raphe nuclei; Red N, red nucleus. Values of scale bars are indicated for each panel.

11 Supplemental Table 1: Serum hormones in male and female mice Male Female Parameter Control SIM1-ERα-KO Control SIM1-ERα-KO T3 (ng/mL) 0.51±0.05 0.4±0.05 0.31±0.05 0.3±0.04 T4 (ng/mL) 34.28±1.65 32.01±2.29 25.46±1.99 26.31±2.15 Norepinephrine (pg/mL) 16002.5±758.55 15298.75±741.29 23611.88±1352.62 23361±2834.8 Epinephrine (pg/mL) 3315.25±695.79 5195.25±1377.85 13324.13±3952.6 16838.25±5029.73 Testosterone (ng/mL) 9.94±5.42 6.32±5.19 ––––––––– ––––––––– 17β-estradiol (pg/ml) ––––––––– ––––––––– 50.86±3.95 62.82±7.07 Leptin ––––––––– ––––––––– 2.15±0.40 5.87±1.60* All data are presented as mean ± SEM. *, P<0.05 between control and SIM1-ERα-KO in t-test.

Supplemental Table 2: Primer sequence (add BAT gene primers)

PCR GenBank Target gene Primer sequences products accession

Androgen receptor (Ar) Ar F TCTTTCAAGGGAGGTTACGC 101bp NM_013476.3 Ar R GAGGACGGGATCTCAAGTGTC

Aromatase Cyp19a1 F ACGTGGATGTGTTGACCCTC 93bp NM_007810.3 Cyp19a1 R TCACAATAGCACTTTCGTCCAG Glutamate decarboxylase 1

(Gad1) Gad1 F AGCCAGACAAGCAGTATGACG 126bp NM_008077.4 Gad1 R GGTTTTCAAATCCCACGGT Glutamic acid decarboxylase 2

(Gad2) Gad2 F ACAGGGCATCCCCGATAT 75bp NM_008078.2 Gad2 R TGTCAACCAGTCTGCTGCTAAT NM_001081081.

Glutaminase (Gls) Gls F GCACAGACATGGTTGGGATAC 126bp 2 Gls R TTTCACCAGTAATTGGGCAGA Glutamate-ammonia ligase

(Glul) Glul F TCATCTTTGGATAGCCCGTTTT 132bp NM_008131.3 Glul R GCCTTGGTGCTGAAGTTGGTA Melanocortin 4 receptor (MC4R) MC4R F GGTCGGAAACCATCGTCA 132bp NM_016977 MC4R R AAAGCAGGCTGCAAATGG

Nitric oxide synthase 1 (Nos1) NOS1 F GATTGGCGTTCGTGATTACTG 69bp NM_008712.2 NOS1 R TCCATTTTCTTGGCTACTTCCT XM_006512632.

Single-minded 1 (SIM1) SIM1 F TGTCTCCCTTTGATGGAT 219bp 1 SIM1 R CATGCACGTGTAGGTACA NM_001271806.

Agouti related peptide (AgRP) AGRP F CGGCCACGAACCTCTGTAG 65bp 1 AGRP R CTCATCCCCTGCCTTTGC

Estrogen receptor alpha (ERa) ERa F AAGAGAGTGCCAGGCTTT 223bp NM_007956.4 ERa R GGAACCGACTTGACGTAG NM_001122899.

Leptin receptor (Lepr) Lepr F GGAGCCATTACCTAAGAACCC 156bp 1 Lepr R TGACATTCACATCCCCGAAG

Neuropeptide (NPY) NPY F TCCGCTCTGCGACACTAC 230bp NM_023456.2 NPY R GGGACAGGCAGACTGGTT Cyclophilin (CYCLO) CYCLO F TGGAGAGCACCAAGACAGACA 66bp NM_011149.2 CYCLO R TGCCGGAGTCGACAATGAT Tripartite motif-containing 25 Trim25 F TGATGTGGCTGTGCATGATA 91bp NM_009546.2 12 (Trim25) Trim25 R AAGACCTGCTCCCCTACGAC NM_001278581. GAGGCCACTGAACATCTTTGTC

Pro-opiomelanocortin (POMC) POMC F 252bp 1 POMC R GCAGAGGCAAACAAGATTGG Uncoupling 1 (UCP1) UCP1F GAGGTGTGGCAGTGTTCATTG 59bp NM_009463 UCP1R GGCTTGCATTCTGACCTTCA CCAAT/enhancer binding protein, alpha (CEBP α) NM_001287514.

CEBP α F GCGCAAGAGCCGAGATAAAG 81bp 1 CEBP α R CGGTCATTGTCACTGGTCAACT E strogen related receptor, alpha (ERRα) ERRα F AGCAAGCCCCGATGGA 105bp NM_007953 ERRα R GAGATGCCTGGGATGCTCTT P eroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PGC-1α) PGC-1α F AACCACACCCACAGGATCAGA 73bp NM_008904.2 PGC-1α R TCTTCGCTTTATTGCTCCATGA P eroxisome proliferator activated receptor alpha (PPAR γ) PPAR γ F CGTACGGCAATGGCTTTATC 55bp NM_011144.6 PPAR γ R AACGGCTTCCTCAGGTTCTT

Nuclear receptor interacting TTCTCAGGACATAATCCTTTAACAT protein 1 (RIP140) RIP140 F TC 72bp NM_173440.2 RIP140 R CGGGCCTCCTCAGTCAAC U ncoupling protein 3 (UCP3) UCP3 F TTTCTGCCACTGGGAGCTT 63bp NM_009464.3 UCP3 R GGCCCTCTTCAGTTGCTCAT A drenergic receptor, beta 3 (ADRB3) ADRB3 F GACTACAGACCATAACCAACGTG 82bp NM_013462 ADRB3 R CCTGGTGGCATTACGAGGA PR domain containing 16 PRDM16 CCACCAGCGAGGACTTCAC (PRDM16) F 107bp NM_027504 PRDM16 GGAGGACTCTCGTAGCTCGAA R

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